1. Field of the Invention
This invention relates to the characterization of the role that glycosylation of the transmembrane glycoprotein E1 of highly virulent Classical Swine Fever Virus (CSFV) strain Brescia plays during infection in the natural host and to the utilization of a strategy for manipulating the pattern of glycosylation for particular E1 glycosylation sites in order to alter CSFV virulence, providing a useful tool in the design and development of CSF live-attenuated vaccines.
2. Description of the Relevant Art
Classical swine fever (CSF) is a highly contagious disease of swine. The etiological agent, CSF virus (CSFV), is a small, enveloped virus with a positive, single-stranded RNA genome and, along with Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), is classified as a member of the genus Pestivirus within the family Flaviridae (Becher et al. 2003. Virology 311: 96-104). The 12.5 kb CSFV genome contains a single open reading frame that encodes a 3898-amino-acid polyprotein and ultimately yields 11 to 12 final cleavage products (NH2—Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B—NS5A-NS5B—COOH) through co- and post-translational processing of the polyprotein by cellular and viral proteases (Rice, C. M. 1996. In: Fundamental Virology, 3rd edition, Knipe et al., eds., Lippincott Raven, Philadelphia, Pa., pages 931-959). Structural components of the CSFV virion include the capsid (C) protein and glycoproteins Erns, E1, and E2. E1 and E2 are anchored to the envelope at their carboxyl termini and Ems loosely associates with the viral envelope (Slater-Handshy et al. 2004. Virology 319: 36-48; Weiland et al. 1990. J. Virol. 64: 3563-3569; Weiland et al. 1999. J. Gen. Virol. 80: 1157-1165). E1 and E2 are type I transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor (Thiel et al. 1991. J. Virol. 65: 4705-4712). E1 has been implicated (Wang et al. 2004. Virology 330: 332-341), along with Erns and E2 (Hulst et al. 1997. J. Gen Virol. 78: 2779-2787), in viral adsorption to host cells. Importantly, modifications introduced into these glycoproteins appear to have an important effect on CSFV virulence (Meyers et al. 1999. J. Virol. 73: 10224-10235; Risatti et al. 2005a. J. Virol. 79: 3787-3796; Risatti et al. 2005. Virology 355: 94-101; Risatti et al. 2005b. Virology 343: 116-127; Van Gennip et al. 2004. J. Virol. 78: 8812-8823).
Glycosylation is one of the most common types of protein modifications. N-linked oligosaccharides are added to specific asparagine residues in the context of the consensus sequence Asn-X-Ser/Thr (Kornfeld and Kornfeld. 1985. Annu. Rev. Biochem. 54: 631-664). According to a glycosylation analysis algorithm (Retrieved from the Internet: cbs.dtu.dk/services/), E1 of the CSFV strain Brescia has three putative N-linked glycosylation sites although this is not confirmed by experimental evidence. Predicted E1 glycosylation sites (at CSFV amino acid residue position N500, N513 and N594) are highly conserved among CSFV isolates and two of them (N513 and N594) are also conserved in other Pestiviruses. However, the significance of viral envelope protein glycosylation in virus replication, pathogenesis, and virulence in the natural host is not completely defined. It has just been recently described that specific removal of certain putative glycosylation sites in Erns and E2 significantly alters the virulence of highly virulent Brescia strain in swine (Fernandez Sainz et al. 2008. Virology (370:122-129); Risatti et al. 2007. J. Vinci. 81: 924-933).
Strategies for controlling disease in the event of a CSFV outbreak include the production of rationally designed live attenuated vaccine CSFV strains. Thus, the effect of modification of glycosylation sites of other of the CSFV virion glycoproteins need to be evaluated. Here, we report the effects of modification of particular predicted E1 glycosylation sites. We used oligonucleotide site-directed mutagenesis of the E1 gene of the highly virulent CSFV strain Brescia to construct a panel of glycosylation mutants. These mutants were evaluated to determine whether the removal of each of these glycosylation sites in the E1 glycoprotein could affect viral infectivity and virulence in swine.